Over the last 13 years, this grant has consisted of two basic aims with the goal of finding a cure for hemophilias A and B, as well as advance the field of gene transfer/therapy in general. In the last 5 years, this grant has supported 20 publications including Nature Biotechnology and two recent Nature publications. Our aims have focused on developing and testing the following tools for these goals: (1) new adeno-associated virus (AAV) vectors and gene transfer approaches; and (2) novel DNA plasmids vectors while also investigating the mechanism of why some episomal plasmids exhibit transcriptional persistence in vivo. Our work has provided the preclinical data that has supported two hemophilia AAV-based gene transfer trials. While these trials have begun to show therapeutic success in factor IX (FIX) deficiency, significant limitations and hurdles towards a cure remain. Our newer episomal plasmids called minicircles and mini-intronic plasmids have been widely distributed and used by various academic and pharmaceutical laboratories for biological discovery and therapeutic development. Discoveries explaining the chromatin/transcriptional mechanisms that define their ability to persistently express or become silenced in quiescent tissues have provided additional insight in the design of new vector systems. The rise in popularity of gene targeting can be attributed in large part to the development of readily engineered and easy-to-use site-specific endonucleases. These can induce gene disruption by cleaving the genome at a locus of choice, or can induce gene correction, insertion or tagging when a suitable donor sequence is provided. Even if transient expression is accomplished - a non- trivial challenge - the use of endonucleases raises concerns including substantial off-target cleavage and off- target integration of the transgene vector and the endonuclease vector (if DNA-based). We have devised a site-specific gene targeting application for the treatment of hemophilia that does not rely on the use of endonucleases. We also avoid using vector-borne promoters so that even in the event of off-target integration, no neighboring gene activation would occur decreasing the risk of oncogenicity. This platform could potentially be used to cure genetic disorders with a single administration of a rAAV vector. In this renewal, we propose studies to: (1) Further develop a promoterless AAV-based strategy to efficaciously and safely target a hFIX coding sequence into endogenous specific genomic regions (e.g. albumin locus) without disrupting endogenous liver gene expression, and without the need for any accessory protein (e.g. nuclease). (2) Define new mechanisms of mammalian transcription using our novel plasmid systems. (3) Develop enhanced expression cassette designs using mini-intronic and minicircle-DNA vector technology, and test them for enhanced expression in plasmids and rAAV vectors. In summary, our studies will contribute to new knowledge in fundamental areas of biomedical science, and provide better viral vectors and DNA expression plasmid reagents for biological discovery and clinical therapeutics, with application to any vector system.